本文關(guān)鍵詞：大鼠骨髓間充質(zhì)干細胞分泌因子對Ⅳ型變態(tài)反應(yīng)的影響　出處：《青島大學》2013年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 骨髓間充質(zhì)干細胞 分泌因子 淋巴細胞 巨噬細胞 接觸性皮炎

【摘要】：目的：觀察骨髓間充質(zhì)干細胞分泌因子對遲發(fā)型超敏反應(yīng)的影響,并初步探討其作用機制,為骨髓間充質(zhì)干細胞分泌因子在臨床上用于過敏性疾病的治療提供一定的理論依據(jù)。 方法：體內(nèi)試驗：①分離培養(yǎng)大鼠骨髓間充質(zhì)干細胞(Bone marrow mesenchymal stem cells, BMSCs),貼壁法純化,收集第3代細胞融合達85%以上的上清液,獲取干細胞分泌因子。②制備小鼠接觸性皮炎模型,觀察干細胞分泌因子對小鼠耳廓腫脹的抑制作用及胸腺指數(shù)和脾臟指數(shù)的影響。③MTT比色法反映分泌因子對小鼠脾淋巴細胞增殖的影響。④采用酶聯(lián)免疫吸附測定法(ELISA法)檢測分泌因子對血清IL-2、IFN-γ細胞因子水平的影響。體外實驗：①分離培養(yǎng)大鼠BMSCs,貼壁法純化,收集第三代細胞融合達85%以上的BMSCs上清液,獲取干細胞分泌因子。②無菌獲取小鼠胸腺、脾臟,制備單細胞懸液,MTT比色法觀察高、中、低劑量組干細胞分泌因子對ConA誘導的淋巴細胞增殖的影響；③分離培養(yǎng)大鼠腹腔巨噬細胞,中性紅法檢測干細胞分泌因子對脂多糖(LPS)刺激后巨噬細胞吞噬活性的影響。 結(jié)果體內(nèi)試驗：①與對照組相比,干細胞分泌因子和地塞米松(DEX)均能夠明顯減輕模型小鼠耳廓腫脹程度,降低胸腺、脾臟指數(shù),抑制脾淋巴細胞的增殖(P＜0.05),但分泌因子的抑制作用弱于DEX (P0.05)。②BMSCs分泌因子與DEX均能夠顯著降低血清中IL-2、IFN-γ水平(P＜0.05)。體外實驗：①與對照組相比,細胞分泌因子高、中劑量和DEX對ConA誘導的小鼠胸腺淋巴細胞增殖均有抑制作用(P＜0.05),高劑量組的抑制作用強于中劑量組(P＜0.05),弱于DEX組(P＜0.05)；分泌因子高劑量組和DEX組對ConA誘導的小鼠脾臟淋巴細胞增殖亦有抑制作用(P＜0.05)。②分泌因子能夠減弱經(jīng)LPS刺激后巨噬細胞吞噬中性紅的能力(P＜0.05)。 結(jié)論骨髓間充質(zhì)干細胞分泌因子具有抗遲發(fā)型超敏反應(yīng)的作用,其抑制特異性T淋巴細胞增殖,降低淋巴細胞分泌IL-2、IFN-γ細胞因子水平,降低活化的巨噬細胞吞噬功能是其發(fā)揮作用的機制之一。
[Abstract]:Objective: To observe the effect of bone marrow mesenchymal stem cells secretory factor on delayed type hypersensitivity, and to explore its mechanism of action, providing a theoretical basis for the clinical application of bone marrow mesenchymal stem cells secretory factor in allergic diseases.
Methods: in vivo experiment: the isolation and culture of rat bone marrow mesenchymal stem cells (Bone marrow mesenchymal stem cells, BMSCs), purified by attachment method, collect the third generation of more than 85% of the supernatant of cell fusion, stem cell factor secretion. Contact dermatitis mouse model by the system, observe the effect of stem cell factor secretion inhibition effect on mouse ear swelling and thymus index and spleen index. The MTT colorimetric method to reflect the influence of cytokines on the proliferation of mouse spleen lymphocytes. 4 by enzyme-linked immunosorbent assay (ELISA) detection of secreted IL-2 clear of blood, the impact factor level of IFN- gamma cells. In vitro: 1. Isolation and culture rat BMSCs, purified by attachment method, collect the third generation of BMSCs was more than 85% cell fusion, stem cell factor secretion. The Sterility from mouse thymus, spleen, preparation of single cell suspension, MTT colorimetric method view The effects of stem cell secretory factors on ConA induced lymphocyte proliferation in rats were observed. (3) the peritoneal macrophages were isolated and cultured, and the effects of stem cell secretory factors on phagocytosis of macrophages stimulated by lipopolysaccharide (LPS) were detected by neutral red method.
The results of experiment in vivo: compared with control group, stem cell factor secretion and dexamethasone (DEX) can significantly reduce the mice ear swelling, reduce thymus, spleen index, inhibit the proliferation of spleen lymphocytes (P < 0.05), but the inhibitory effect of secretory factors is weaker than that of DEX (P0.05). The secretion of BMSCs and factor DEX can significantly reduce the serum IL-2, IFN- levels (P < 0.05). In vitro experiment: compared with control group, cell factor secretion inhibition in high dose and DEX on mouse thymus lymphocyte proliferation were induced by ConA (P < 0.05), a strong inhibiting effect of high dose group in middle dose group (P < 0.05), weaker than the DEX group (P < 0.05); secreted in high dose group and DEX group also have inhibitory effect on mice spleen lymphocyte proliferation induced by ConA (P < 0.05). The secreted factor can decrease after LPS stimulation of macrophage phagocytosis of neutral red can Force (P < 0.05).
Conclusion the secretion factor of bone marrow mesenchymal stem cells has the effect of anti delayed hypersensitivity. It inhibits the proliferation of specific T lymphocytes, lowers the secretion of IL-2 and IFN- gamma cytokines by lymphocytes, and reduces the phagocytosis function of activated macrophages, which is one of the mechanisms of its action.

【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R758.2

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